Methods for treatment of clostridium difficile infections

ABSTRACT

In this study, we capitalized on the antimicrobial property and low oral bioavailability of known salicylanilide anthelmintics (closantel, rafoxanide, niclosamide, oxyclozanide) to target the gut pathogen. The anthelmintics displayed excellent potency against  C. difficile  strains 630 and 4118 (with MIC values as low as 0.06-0.13 μg/mL for rafoxanide) via a membrane depolarization mechanism. Interestingly, closantel, rafoxanide and compound 8 were bactericidal against logarithmic- and stationary-phase cultures of the BI/NAP1/027 strain 4118. Further evaluation of the salicylanilides showed their preferential activity against Gram-positive over Gram-negative bacteria. Moreover, the salicylanilides were non-hemolytic and were non-toxic to mammalian cell lines HepG2 and HEK 293T/17 within the range of their in vitro MICs and MBCs. The salicylanilide anthelmintics exhibit desirable bactericidal and pharmacokinetic properties and are amenable to repositioning as anti- C. difficile  agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application and claims the benefit ofpriority to U.S. Ser. No. 16/317,914, filed Jan. 15, 2019, which is anational stage application filed under 35 U.S.C § 371 from InternationalApplication Serial No. PCT/US2017/042056, filed on Jul. 14, 2017, andpublished as WO 2018/013890 on Jan. 18, 2018, which claims the benefitof priority to U.S. provisional application Ser. No. 62/362.675, filedon Jul. 15, 2016, the disclosures of which are incorporated herein byreference in their entirety.

BACKGROUND

Clostridium difficile infections (CDI) has plagued nearly half a millionAmericans that resulted in 29,300 deaths in 2011,¹ and the propensity ofnosocomial CDI recurrence has been observed in up to 50% of patients.²The growing epidemic of CDI has been largely attributed to the emergenceof the hypervirulent strain BI/NAP1/027,³⁻⁵ coupled with the paucity oftherapeutics that specifically target the gram-positive, spore-formingbacillus as well as, prevent the recrudescence of the disease. Althoughcurrent treatment options (metronidazole and vancomycin) are still ableto manage moderate cases of CDI, the escalating rates of fulminant andrecurrent infections pose a significant threat that warrant immediateattention. Fidaxomicin is a non-absorbed oral macrocyclic antibioticthat was recently approved by the FDA for the treatment of CDI. Itdemonstrated similar rates of clinical cure as vancomycin^(6,7) andsignificantly lowered the rate of recurrence of non-NAP1-associatedinfections⁶—a finding that is attributable to its high selectivityagainst C. difficile ^(8,9) and its ability to inhibit toxin and sporeproduction in the offending pathogen.^(10,11) However, there was nodifference in outcomes observed for patients that were infected with thehypervirulent BI/NAP1/027 strain.⁸ Although resistance is not widespreadas of yet, C. difficile strains with reduced susceptibility tometronidazole, vancomycin or fidaxomicin have already been noted.¹²⁻¹⁴

The persistence of CDI is alarming in its breadth and points to thepressing need to identify effective treatment options. As a result, thescientific community has risen to the challenge of developingalternative small molecule and biotherapeutic strategies to combat theinfectious malady.¹⁵ It is evident that anti-difficile agents with loworal bioavailability (to localize the drug at the site of infection) anda narrow antimicrobial spectrum (to minimize collateral damage to theresident gastrointestinal microbiome) are preferable. Hypervirulent C.difficile isolates have been shown to produce robust amounts of lethaltoxins (TcdA and TcdB) and spores primarily during the stationary phaseof growth.⁴ This sets an impediment because quiescent stationary-phasecells are especially resilient to antimicrobial chemotherapy.¹⁶ Anemerging strategy to combat refractory dormant C. difficile is to targetthe vulnerability of its membrane. The clinical relevance of suchconcept lies in the essentiality of the microbial membrane in bothmetabolizing and non-growing cells, and the associated multifactorialmechanism of action that could limit the likelihood of bacteria todevelop resistance.¹⁷ Indeed, membrane-active agents have demonstratedpotential in eliminating quiescent C. difficile cells, whichsubsequently led to a substantial decrease in toxin production andsporulation.^(16,18,19)

The salicylanilides have been reported to exhibit antimicrobialproperties^(20,21) albeit they are chiefly exploited as antiparasiticagents. Closantel (1), rafoxanide (2), niclosamide (3) and oxyclozanide(4) represent four of the widely used salicylanilide anthelmintics (FIG.1). Iclosamide is an FDA-approved drug for the treatment of tapeworminfections, while the other three are marketed as veterinary drugs forliver fluke/roundworm infections in ruminants.²² The exact antibacterialmode of action of salicylanilides is not well defined but is thought toinvolve dissipation of the (trans)membrane potential or the protonmotive force (pmf). The pmf modulates the spatial organization ofmorphogenetic proteins²³ as well as ATP homeostasis that is vital forbacterial survival.²⁴ These functions of the pmf offer an explanationfor the effects observed with certain membrane-active compounds, albeitdepletion of which does not always result to cell death in manybacterial pathogens.²⁵ The potential use of salicylanilides asantimicrobials has drawn considerable interest as exemplified by recentstudies demonstrating the anti-staphylococcal properties of closantel,niclosamide and oxyclozanide.^(26,27)

A limiting aspect is the low oral bioavailability of salicylanilides,which may render them ineffective in treating systemic infections. Forinstance, niclosamide was found to be only partially absorbed from theGI tract (with a maximal serum concentration ranging from 0.25 to 6μg/mL after oral administration to human volunteers) and was also poorlydistributed to tissues.²⁸ Closantel, rafoxanide and oxyclozanideexhibited similar pharmacokinetic (PK) attributes and were minimallymetabolized and mostly excreted unchanged (up to ˜90% for closantel) inthe feces in ruminants.²²

SUMMARY

The invention provides, in various embodiments, a method of treatment ofa Clostridium difficile infection in a mammal, comprising administeringto the mammal an effective dose of a compound of formula (I)

wherein X is halo or H, provided at least one X is halo, wherein thering bearing X is optionally further substituted with halo;

wherein Ar is phenyl, benzyl, phenethyl, biphenyl, benzyhydryl,phenoxyphenyl, naphthyl, or indanyl, any of which can be unsubstitutedor independently substituted with one or more halo, (C1-C4)alkyl, cyano,or nitro groups.

More specifically, X can be chloro or iodo. More specifically, Ar can bephenyl, phenethyl, or phenoxyphenyl, any of which can be substitutedwith halo or (C1-C4)alkyl or both.

For instance, the compound of formula (I) can be any one of compoundsclosantel (1), rafoxanide (2), niclosanide (3), oxyclozanide (4), or ofany one of a compound of formula (5a), (5e), (5f), (5g), (6a), (7a),(7b), (7c), (7d), (7e), (7f), (7g), (7h), (7i), or (8).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structures of salicylanilide anthelmintics useful for practiceof a method of the invention.

FIG. 2. Structures of salicylanilide analogues useful for practice of amethod of the invention.

FIG. 3. Time-kill kinetics against stationary-phase cultures ofBI/NAP1/027 strain 4118. Various concentrations of A) closantel, B)rafoxanide, C) compound 8, and D) metronidazole or vancomycin are shown.Data plotted as mean log₁₀ cfu/mL±s. d. versus time in h (n=2).

DETAILED DESCRIPTION

Prolonged use of broad-spectrum antibiotics disrupts the indigenous gutmicrobiota, which consequently enables toxigenic Clostridium difficilespecies to proliferate and cause infection. The burden of C. difficileinfections was exacerbated with the outbreak of hypervirulentBI/NAP1/027 strains that produce copious amounts of enterotoxins andspores. In recent past, membrane-active agents have generated a surge ofinterest due to their bactericidal property with a low propensity forresistance.

We show that the salicylanilide derivatives efficiently inhibited thegrowth of C. difficile via membrane depolarization, and moreimportantly, killed both logarithmic- and stationary-phase cells in aconcentration-dependent manner. The bactericidal property againstquiescent C. difficile could in principle lower the production of toxinsand spores, which would in turn mitigate disease severity andrecurrence.

We initially tested the known anthelmintics closantel, rafoxanide,niclosamide and oxyclozanide for their activities against C. difficilestrains 630 (CD630, ATCC BAA-1382) and 4118 (CD4118, ATCC BAA-1870).CD630 is a virulent, multidrug-resistant strain whose genome has beencompletely sequenced,²⁹ while CD4118 is a BI/NAP1/027 hypervirulentpathogen. All four salicylanilides displayed excellent potency with MICvalues as low as 0.06-0.13 μg/mL for rafoxanide (Table 1). Incomparison, metronidazole had an MIC value of 0.25 μg/mL, whereas thatof vancomycin was significantly higher at 1-2 μg/mL (Table 1). In orderto ascertain that the observed activity of the salicylanilides occursthrough dissipation of the bacterial membrane potential, we preparedanalogues 5 and 6 (FIG. 2) as previously described,³⁰ and evaluatedtheir growth inhibitory activity against CD630 and CD4118. We haveearlier delineated the structural features that are necessary forprotonophoric activity of salicylanilides, requiring both a dissociablephenolic OH group and an amide proton that forms an intramolecularhydrogen bond to maintain hydrophobicity and stabilize the anionic formof the molecule.³⁰ The MIC values that were determined for 5 and 6 areconsistent with a membrane depolarization mechanism as the compoundsdevoid of protonophoric activity [i.e. analogues that lack either theweakly acidic OH (5b, 5c, 5h, 5i, 6b and 6c) or the amide proton (5d)]were inactive, whereas protonophores 5a, 5e, 5f, 5g, and 6a exhibitedhigh in vitro potency against CD630 and CD4118 (Table 1). Encouraged bythese results, we explored several other derivatives, which harbor thediidosalicylate moiety coupled to varying substituents includingbiphenyl (7a), halogenated mono-aryl rings (7b-d), a fused-ringfluorenyl core (7e) and the more flexible ethylbenzenes (7f-i).Compounds 7a-i demonstrated low MIC values (≤2 μg/mL), except for theortho-chloro analogue 7c, which showed reduced activity against CD630and CD4118 (MIC=8 μg/mL). Replacement of the diiodosalicylate with itsdichloro congener (compound 8) resulted in a 4-fold enhancement ofpotency relative to 5g and metronidazole, and ˜32-fold improvement ofactivity over vancomycin (Table 1).

TABLE 1 MIC against Clostridium difficile strains 630 and 4118. Allminimum inhibitory concentration (MIC) values are expressed in pg/mL.MIC Cmpd C. difficile 630 C. difficile 4118 Closantel 0.13 0.25Rafoxanide 0.06 0.13 Niclosamide 1 4 Oxyclozanide 0.5 1 5a 0.5 15b >32 >32 5c >32 >32 5d >32 >32 5e 0.13 0.13 5f 0.13 0.25 5g 0.13 0.255h >32 >32 5i >32 >32 6a 0.5 0.5 6b >32 >32 6c >32 >32 7a 0.5 1 7b 0.250.5 7c 8 8 7d 0.13 0.25 7e 0.25 0.5 7f 2 2 7g 1 1 7h 0.25 0.5 7i 0.250.5 8 ≤0.03 0.06 Metronidazoe 0.25 0.25 Vancomycin 1 2The foregoing observations led us to probe other ionophores such astropolones and β-carbolines as well as other structurally relatedcompounds lacking the salicylanilide moiety; however, none of these werefound to be active against CD630 and CD4118 (MIC>32 μg/mL).

Salicylanilides are Bactericidal Against Logarithmic- andStationary-Phase Cultures

The superb growth inhibitory potency exhibited by the salicylanilidesspurred us to further investigate their bactericidal activities againstC. difficile. Although ionophores are known to dissipate the pmf that iscrucial for bacterial energy metabolism, they do not always displaybactericidal activity.^(25,27) We were particularly interested indetermining the cidal effect on stationary-phase C. difficile cells,because these quiescent cells are the primary producer of toxins andspores that contribute to the severity and recurrence of CDI.⁴ Weselected the more potent compounds (closantel, rafoxanide and 8) andassayed them for minimum bactericidal concentration (MBC, defined as thelowest concentration of the antibacterial agent required to kill≥99.9%of the initial inoculum) against growing and non-growing cells of theBI/NAP1/027 pathogen CD4118. As shown in Table 2, all three compoundsdisplayed bactericidal activities against both logarithmic- andstationary-phase cells of CD4118 at concentrations close to their MICvalues. The MBC_(log) values of the protonophores were determined to be0.25-2 μg/mL (˜4 to 8-fold greater than their respective MIC values).Significantly, the salicylanilides retained bactericidal activitiesagainst dormant stationary-phase C. difficile cells, in stark contrastto metronidazole and vancomycin, which did not result in ≥3-logreduction of CD4118 cells at 32 μg/mL (Table 2).

Next, we determined the time-kill kinetics of closantel, rafoxanide and8 (at 1×, 4×, and 16× their respective MICs) against stationary-phasecultures of CD4118. As depicted in FIG. 3, all three salicylanilidesshowed a concentration-dependent mode of killing of the quiescent cells.At 16× the MIC of each protonophore, rafoxanide (at 2 μg/mL)eradicated >99.9% of viable cells in 6 h (FIG. 3b ), while closantel (at4 μg/mL) and compound 8 (at 1 μg/mL) achieved a similar potency in 24 h(FIGS. 3a and 3c ). At four-fold lower concentrations (i.e. 4×MIC),rafoxanide caused a 2.7-log decrease in the number of CFUs in 24 h,comparable to those of closantel and 8, which reduced bacterial cellviability by 2.2- and 2.4-log, respectively. In comparison, neithermetronidazole (at 4 μg/mL) nor vancomycin (at 32 μg/mL) reached ≥3-logkilling of CD4118 stationary-phase cells, even at 16× their respectiveMIC values (FIG. 3d ). The rapid bactericidal property demonstrated byclosantel, rafoxanide and 8 is a significant finding because quiescentC. difficile cells are notoriously recalcitrant to antibiotic-mediatedkilling.¹⁶ We surmise that the cidal effect of such protonophores onstationary-phase C. difficile cells would ameliorate the effect of toxinproduction and spore formation, similar to what was observed with othermembrane-active compounds.¹⁶

TABLE 2 In vitro activity against Clostridium difficile strain 4118.Cmpd MIC MBC_(log) MBC_(stat) Closantel 0.25 2 4 Rafoxanide 0.13 0.5 1 80.06 0.25 1 Metronidazole 0.25 >32 >32 Vancomycin 2 8 >32 Abbreviations:MIC, minimum inhibitory concentration; MBC_(log), minimum bactericidalconcentration for logarithmic-phase cells; MBC_(stat), minimumbactericidal concentration for stationary-phase cells, All MIC and MBCvalues are expressed in μg/mL.

Salicylanilides Mainly Target Gram-Positive Bacteria

In an effort to assess the ant bacterial spectrum of protonophores weevaluated representative compounds (closantel, rafoxanide, 6a, 7b, 8)against a panel of aerobic and anaerobic organisms. All five agents weregenerally more selective against Gram-positive bacteria, displaying highpotency against B. subtilis ATCC 6051, S. aureus RN4220 and S.epidermidis 1457 (MIC≤0.25 μg/mL;) and modest activity against otheranaerobic clostridial species C. sporogenes ATCC 15579 and C.clostridioforme ATCC 25537 (MIC=1-16 μg/mL). By comparison, thecompounds were ineffective against aerobic Gram-negative bacteria(MIC≥32 μg/mL against A. baumannii M2 and P. aeruginosa PAO1) and hadmodest MIC values of ≥4 μg/mL against gut commensals B. thetaiotaomicronATCC 29148, P. distasonis ATCC 8503 and P. nigrescens ATCC 33563. Theseresults are consistent with those of niclosamide and oxyclozanide, whichwere shown to primarily target Gram-positive bacteria.²⁷ Compound 5i,which does not possess protonophonc activity,⁶⁰ lacked antibacterialactivity whereas metronidazole and vancomycin mainly targeted anaerobicbacteria and Gram-positive organisms, respectively. The complexmultilayered cell envelopes of Gram-negative organisms impose apermeability barrier to microbial agents and most likely account for thediminished potency observed for the salicylanilide molecules. Of note,rafoxanide and 8 had MIC values of ≤0.13 μg/mL for C. difficile, whichrendered ≥32-fold selectivity over the Gram-negative gut commensals thatwere tested.

In Vitro Cytotoxicity and Hemolytic Activity of Salicylanilides

Although the salicylanilides have been used extensively in veterinarymedicine, there is little information available concerning theirbiological effects on humans, except for niclosamide, which isFDA-approved for treatment of intestinal cestode infections. In order togauge potential cytotoxicity of the salicylanilides, hemolysis usingsheep erythrocytes and MTS³³ assay using two human cell lines (livercarcinoma HepG2 and embryonic kidney HEK 293T/17) were performed. Asignificant finding was that the salicylanilides (closantel, rafoxanide,niclosamide, oxyclozanide and compound 8) did not cause rupture of redblood cells when tested at 32 μg/mL. However, treatment of human celllines with niclosamide led to a significant decrease in viability evenat a low concentration of 0.125 μg/mL. Despite its high in vitrocytotoxicity, niclosamide is considered a “safe drug” because of itsminimal absorption from the GI tract and high plasma protein binding,²⁸thus sparing the host cells from its uncoupling property. An intriguingobservation was the comparably lower in vitro toxicities of compound 8and the veterinary drugs (closantel, rafoxanide, oxyclozanide) towardHepG2 and HEK 293T/17. Both closantel and rafoxanide had no apparenteffect on mammalian cell viability even at a concentration of 8 μg/mL,which is ≥32-fold higher than their corresponding MIC values against C.difficile (Table 1). These results do not guarantee drug safety(relative to niclosamide) but nevertheless indicate the potential forrepositioning of the veterinary anthelmintics as human drugs.

A common cause of antibiotic failure is the inadequate penetration ofthe target infection site. In the case of CDI, it is imperative that theactive drug achieves therapeutic levels in the colon to repress oreliminate the outgrowth of toxigenic C. difficile. This places thesalicylanilide anthelmintics at a definite advantage; their low oralbioavailability and high fecal excretion (as observed in ruminants andhumans)^(22,28) would in theory result in adequate gut concentrationsnecessary to disarm the target pathogen. A substantial feature of thesalicylanilides (as we have shown for closantel, rafoxanide and 8) istheir bactericidal activity against stationary-phase cultures ofhypervirulent C. difficile—a property that is not exhibited by manyantibiotics including metronidazole and vancomycin.¹⁶ Killing of dormantand hypervirulent C. difficile could likely suppress toxin productionand inhibit sporulation, which in principle would lead to an improvedsustained response and reduced recurrence rate. The clinical potentialof membrane-active agents is demonstrated by daptomycin and telavancin,which function through permeabilization/depolarization of bacterialmembranes and are FDA-approved to treat complicated skin and skinstructure infections.^(34,35) Our results exemplify notable attributesof the salicylanilide anthelmintics and demonstrate their potential forrepurposing as anti-Clostridium difficile agents. Work is ongoing in ourlaboratory to exploit the salicylanilides as alternative therapies tocombat CDI.

DOCUMENTS CITED

-   1 Lessa, F. C. et al. Burden of Clostridium difficile infection in    the United States. N. Engl. J. Med. 372, 825-834, (2015).-   2 Aslam, S., Hamill, R. J. & Mosher, D. M. Treatment of Clostridium    difficile-associated disease: old therapies and new strategies.    Lancet Infect. Dis. 5, 549-557, (2005).-   3 Loo, V. G. et al. A predominantly clonal multi-institutional    outbreak of Clostridium difficile—associated diarrhea with high    morbidity and mortality. N. Engl. J. Med. 353, 2442-2449, (2005).-   4 Merrigan, M. et al. Human hypervirulent Clostridium difficile    strains exhibit increased sporulation as well as robust toxin    production. J. Bacteriol. 192, 4904-4911, (2010).-   5 Kelly, C. P. & LaMont, J. T. Clostridium difficile—more difficult    than ever. N. Engl. J. Med. 359, 1932-1940, (2008).-   6 Louie, T. J. et al. Fidaxomicin versus vancomycin for Clostridium    difficile infection. N. Engl. J. Med. 364, 422-431, (2011).-   7 Cornely, O. A., Miller, M. A., Louie, T. J., Crook, C. W. &    Gorbach, S. L. Treatment of first recurrence of Clostridium    difficile infection: fidaxomicin versus vancomycin. Clin. Infect.    Dis. 55 Suppl 2, S154-161, (2012).-   8 Louie, T. J., Emery, J., Krulicki, W., Byrne, B. & Mah, M. OPT-80    eliminates Clostridium difficile and is sparing of bacteroides    species during treatment of C. difficile infection. Antimicrob.    Agents Chemother. 53 261-263, (2009).-   9 Credito, K. L. & Appelbaum, P. C. Activity of OPT-80, a novel    macrocycle, compared with these of eight other agents against    selected anaerobic species. Antimicrob. Agents Chemother. 48,    4430-4434, (2004).-   10 Babakhani, F. et al. Fidaxomicin inhibits spore production in    Clostridium difficile. Clin. Infect. Dis. 55 Suppl 2, S162-169,    (2012).-   11 Babakhani, F. et al. Fidaxomicin inhibits toxin production in    Clostridium difficile. J. Antimicrob. Chemother 68 515-522, (2013).-   12 Pelaez, T. et al. Metronidazole resistance in Clostridium    difficile is heterogeneous. J. Clin. Microbiol. 46, 3028-3032,    (2008).-   13 Snydman, D. R., Jacobus, N. V. & McDermott, L. A. Activity of a    novel cyclic lipopeptide, CB-183,315, against resistant Clostridium    difficile and other Gram-positive aerobic and anaerobic intestinal    pathogens. Antimicrob. Agents Chemother. 56, 3448-3452, (2012).-   14 Goldstein, E. J. et al. Comparative susceptibilities to    fidaxomicin (OPT-80) of isolates collected at baseline, recurrence,    and failure from patients in two phase III trials of fidaxomicin    against Clostridium difficile infection. Antimicrob. Agents    Chemother. 55, 5194-5199, (2011).-   15 Jarrad, A. M., Karoli, T., Blaskovich, M. A., Lyras, D. &    Cooper, M. A. Clostridium difficile drug pipeline: challenges in    discovery and development of new agents. J. Med. Chem. 58,    5164-5185, (2015).-   16 Wu, X., Cherian, P. T., Lee, R. E. & Hurdle, J. G. The membrane    as a target for controlling hypervirulent Clostridium difficile    infections. J. Antimicrob. Chemother. 68, 806-815, (2013).-   17 Hurdle, J. G., O'Neill, A. J., Chopra, I. & Lee, R. E. Targeting    bacterial membrane function: an underexploited mechanism for    treating persistent infections. Nat. Rev. Microbiol. 9, 62-75,    (2011).-   18 Bouillaut, L. et al. Effects of surotomycin on Clostridium    difficile viability and toxin production in vitro. Antimicrob.    Agents Chemother. 59, 4199-4205, (2015).-   19 Hurdle, J. G., Heathcott, A. E., Yang, L., Yan, B. & Lee, R. E.    Reutericyclin and related analogues kill stationary phase    Clostridium difficile at achievable colonic concentrations. J.    Antimicrob. Chemother. 66, 1773-1776, (2011).-   20 Macielag, M. J. et al. Substituted salicylanilides as inhibitors    of two-component regulatory systems in bacteria. J. Med. Chem. 41,    2939-2945, (1998).-   21 Pauk, K. et al. New derivatives of salicylamides: Preparation and    antimicrobial activity against various bacterial species. Bioorg.    Med. Chem. 21, 6574-6581, (2013).-   22 Swan, G. E. The pharmacology of halogenated salicylanilides and    their anthelmintic use in animals. J. S. Afr. Vet. Assoc. 70, 61-70    (1999).-   23 Strahl, H & Hamoen, L. W. Membrane potential is important for    bacterial cell division. Proc. Natl. Acad. Sci. U.S.A. 107,    12281-12286, (2010).-   24 Rao, S. P., Alonso, S., Rand, L., Dick, T. & Pethe, K. The    protonmotive force is required for maintaining ATP homeostasis and    viability of hypoxic, nonreplicating Mycobacterium tuberculosis.    Proc. Natl. Acad. Sci. U.S.A. 105, 11945-11950, (2008).-   25 Tempelaars, M. H., Rodrigues, S. & Abee, T. Comparative analysis    of antimicrobial activities of valinomycin and cereulide, the    Bacillus cereus emetic toxin. Appl. Environ. Microbiol. 77,    2755-2762, (2011).-   26 Rajamuthiah, R. et al. Whole animal automated platform for drug    discovery against multi-drug resistant Staphylococcus aureus. PLoS    One 9, e89189, (2014).-   27 Rajamuthiah, R. et al. Repurposing salicylanilide anthelmintic    drugs to combat drug resistant Staphylococcus aureus. PLoS One 10,    e0124595, (2015).-   28 Andrews, P., Thyssen, J. & Lorke, D. The biology and toxicology    of molluscicides, Bayluscide. Phamacol. Ther. 19, 245-295 (1983).-   29 Riedel, T. et al. Genome resequencing of the virulent and    multidrug-resistant reference strain Clostridium difficile 630.    Genome Announc. 3, (2015).-   30 Gooyit, M., Tricoche, N., Lustigman, S. & Janda, K. D. Dual    protonophore-chitinase inhibitors dramatically affect O. volvulus    molting. J. Med. Chem. 57, 5792-5799, (2014).-   31 Gooyit, M. et al. Onchocerca volvulus molting inhibitors    identified through scaffold hopping. ACS Infect. Dis. 1, 198-202    (2015).-   32 Gooyit, M., Tricoche, N., Javor, S., Lustigman, S. & Janda, K. D.    Exploiting the polypharmacology of β-carbolines to disrupt O.    volvulus molting. ACS Med. Chem. Lett. 6, 339-343, (2015).-   33 Malich, G., Markovic, B. & Winder, C. The sensitivity and    specificity of the MTS tetrazolium assay for detecting the in vitro    cytotoxicity of 20 chemicals using human cell lines. Toxicology 124,    179-192 (1997).-   34 Hawkey, P. M. Pre-clinical experience with daptomycin J.    Antimicrob. Chemother. 62 Suppl 3, iii7-14, (2008).-   35 Zhanel, G. G. et al. New lipoglycopeptides: a comparative review    of dalbavancin, oritavancin and telavancin. Drugs 70, 859-836,    (2010).

All patents and publications referred to herein are incorporated byreference herein to the same extent as if each individual publicationwas specifically and individually indicated to be incorporated byreference in its entirety.

EXAMPLES

-   Bacterial strains. Clostridium difficile 630 (ATCC® BAA-1382-FZ™),    Clostridium difficile 4118 (ATCC® BAA-1870™), Clostridium sporogenes    (ATCC® 15579™), Clostridium clostridioforme (ATCC® 25537™),    Bactercides thetaiotaomicron (ATCC® 29148™), Parabacteroides    distasonis (ATCC® 8503™) Prevotella nigrescens (ATCC® 33563™), and    Bacillus subtilis (ATCC® 6051™) were purchased from ATCC (Manassas,    Va. USA). Pseudomonas aeruginosa PAO1 was provided by Dr. Kendra    Rumbaugh.-   Bacterial culture. Clostridium species were routinely cultured    either on blood agar base II plates with 5% sheep blood (Teknova),    or in brain-heart infusion broth/agar plates supplemented with 0.5%    yeast extract (BHIS) containing 0.03% L-cysteine. Bacteroides    thetaiotaomicron, Parabacteroides distasonis, and Prevotella    nigrescens were grown on Brucella broth/agar plates supplemented    with hemin (5 μg/mL), vitamin K₁ (1 μg/mL) and 5% lysed horse blood.    Anaerobic bacterial culture was performed in an anaerobic cabinet    (Coy Lab Products) at 37° C. in a reducing anaerobic atmosphere (8%    H₂, 8% CO₂, 84% N₂). All broths and 96-well plates were pre-reduced    (incubated anaerobically overnight) prior to use for anaerobic    culture. Aerobic bacteria were routinely cultured on Mueller-Hinton    broth/agar plates.-   Determination of minimum inhibitory concentration (MIC). All MICs    were determined in 96-well plates using the broth microdilution    method. Two-fold serial dilutions of test compounds were inoculated    with ˜5×10⁵ cfu/mL bacteria. MIC was recorded as the lowest    concentration of the test compound that inhibited visible bacterial    growth after 20-24 h of incubation at 37° C. MIC assays were    performed in duplicate.-   Determination of minimum bactericidal concentration (MBC).    Clostridium difficile strain 4118 was grown to OD₆₀₀˜0.4-0.5    (logarithmic phase) or for 24 h (stationary phase), and thereafter    added to two-fold serial dilutions of test compounds. Cultures were    incubated for 20-24 h at 37° C., and then viable counts were    enumerated on BHIS agar plates. The MBC was determined as the lowest    concentration of the test compound that resulted in ≥3-log reduction    of the initial cell inoculum. MBC measurements were performed in    duplicate.-   Time-kill kinetics assay. Stationary phase cultures of Clostridium    difficile strain 4118 were treated with closantel, rafoxanide,    compound 8 at 1×, 4×, 16×MIC or with metronidazole and vancomycin at    16×MIC. At various time points, sample aliquots were taken and    determined for bacterial viability on BHIS agar plates. Kinetic    experiments were performed in duplicate.-   In vitro cytotoxicity assay. Cell lines Hep G2 [HEPG2] (ATCC®    HB-8065™) and 293T/17 [HEK 293T/17] (ATCC® CRL-11268™) were    purchased from ATCC and cultured according to manufacturer's    instructions. HEPG2 or HEK 293T/17 cells were plated in 96-well    plates, and incubated at 37° C. in a 5% CO₂ humidifying chamber for    24 h. Cells were then treated with test compounds at varying    concentrations, and an MTS assay was performed at 16-h    post-incubation at 37° C. in a 5% CO₂ humidifying chamber, using the    CellTiter 96 aqueous non-radioactive cell proliferation assay kit    (Promega, Madison, Wis., USA) per manufacturer's instructions. MTS    assays were performed in duplicate.-   Hemolysis assay. Sheep red blood cells (Innovative Research, Novi,    Mich., USA) were washed three times with PBS pH 7.4. A 3% cell    suspension in PBS (100 μL) was added to test compounds in PBS (100    μL), and then incubated at 37° C. for 1 h. The plate was centrifuged    at 500×g for 10 min, and supernatants (100 μL) were transferred to a    clean 96-well plate. Hemolysis was determined by measuring    absorbance at 540 nm, with 1% Triton X-100 as the positive control    and 0.5% DMSO in PBS as the negative control. Hemolysis assays were    performed in triplicate.

Tables 3 and 4 provide an indication of the bioactivity of compounds(5i), (6a), (7b), and (8) versus a selection of aerobic and anaerobicbacteria, respectively.

TABLE 3 In vitro activity against select aerobic bacteria MIC^(a)(μg/mL) Clo- Ra- metro- van- strain santel foxanide 5i 6a 7b 8 nidazolecomycin B. subtilis ≤0.03 ≤0.03 >32 0.06 0.06 ≤0.03 >32 0.13 ATCC 6051S. aureus RN4220 0.25 0.25 >32 0.25 0.06 0.13 >32 1 S. epidermidis ≤0.03≤0.03 >32 0.06 ≤0.03 ≤0.03 >32 2 1457 A. baumannii M2 >32 >32 >32 >32 3232 >32 >32 P. aeruginosa >32 >32 >32 >32 >32 >32 >32 >32 PAO1^(a)Performed in duplicate. For clarity, MIC values againstGram-positive and Gram-negative bacteria are shown in blue and red,respectively.

TABLE 4 In vitro activity against select anaerobic bacteria MIC^(a)(μg/mL) strain Closantel Rafoxanide 5i 6a 7b 8 metronidazole vancomycinC. sporogenes 1 1 >32 8 16 4 0.25 2 ATCC 15579 C. clostridioforme 41 >32 4 16 4 0.06 0.5 ATCC 25537 B.thetaiotaomicron >32 >32 >32 >32 >32 >32 1 >32 ATCC 29148 P. distasonis16 8 >32 16 8 4 2 >32 ATCC 8503 P. nigrescens 8 8 >32 8 4 4 2 >32 ATCC33563 ^(a)Performed in duplicate. For clarity, MIC values againstGram-positive and Gram-negative bacteria are shown in blue and red,respectively.

Synthesis and Characterization of Compounds

Closantel (Sigma), rafoxanide (TCI America), niclosamide (Combi-Blocks),oxyclozanide (Sigma), metronidazole (Combi-Blocks), and vancomycinhydrochloride hydrate (Sigma) were used as received.

Compounds 5a-i, 6a-c, 7a-d, 9a-b, 10a-b, 11a-f and 12 were prepared aspreviously described.²⁻⁴ Compounds 7e-i and 8 were synthesized accordingto published procedure.² Briefly. 3,5-diiodosalicylic acid (or3,5-dichlorosalicylic acid, 1 eq) was heated to reflux with SOCl₂ (5 eq)for 7 h, and thereafter concentrated under reduced pressure. Thecorresponding acyl chloride product was precipitated with cold hexanes,filtered and air-dried. Coupling with the respective amine (1 eq) wasperformed in DMF in the presence of DIPEA (3 eq) at rt for 1 h. Allsalicylanilide products were purified by preparative HPLC. Reagents andsolvents were obtained from commercial sources, and reactions werecarried out using technique known to those having ordinary skill in theart.

¹H and ¹³C NMR spectra were recorded on Bruker DRX-600 equipped with a 5mm DCH cryoprobe. Purity of all tested products were generally >95% asassessed by HPLC.

N-(9H-Fluoren-2-yl)-2-hydroxy-3,5-diiodobenzamide (7e). Yield: 40%. ¹HNMR (600 MHz, CDCl₃) δ 3.94 (s, 2H), 7.30-7.34 (m, 1H), 7.39 (t, J=7.3Hz, 1H), 7.44-7.48 (m, 1H), 7.56 (d, J=7.4 Hz, 1H), 7.75-7.81 (m, 2H),7.81 (d. J=1.8 Hz, 1H), 7.89 (s, 1H), 7.98 (s, 1H), 8.20 (d, J=1.8 Hz,1H). ¹³C NMR (151 MHz, CDCl₃) δ 37.2, 80.4, 89.1, 116.9, 118.5, 120.0,120.3, 120.5, 125.2, 127.0, 127.1, 134.3, 134.8, 139.9, 141.0, 143.4,144.6, 151.1, 160.5, 166.4. HRMS-ESI (m/z): [M+H]⁺ calcd forC₂₀H₁₄I₂NO₂, 553.9114; found, 553. 9110.

N-(2Chlorophenethyl)-2-hydroxy-3,5-diiodobenzamide (7f). Yield: 49%. ¹HNMR (500 MHz, DMSO-d₆) δ 2.99 (t, J=7.2 Hz, 2H), 3.54 (q, J=7.0 Hz, 2H),7.24-7.31 (m, 2H), 7.34 (dd, J=2.1, 7.2 Hz, 1H), 7.44 (dd, J=1.9, 7.3Hz, 1H), 8.16 (d, J=1.9 Hz, 1H), 8.18 (d. J=1.9 Hz, 1H), 9.26 (t, J=5.6Hz, 1H). ¹³C NMR (151 MHz, DMSO) δ 32.3, 81.4, 88.8, 116.2, 127.4,128.4, 129.3, 131.2, 133.2, 135.1, 136.4, 149.4, 159.8, 168.1. HRMS-ESI(m/z): [M+H]⁺ calcd for C₁₅H₁₃ClI₂NO₂, 527.8719; found, 527. 8706.

N-(3-Chlorophenethyl)-2-hydroxy-3,5-diiodobenzamide (7g). Yield: 53%. ¹HNMR (500 MHz, DMSO-d₆) δ 2.87 (t, J=7.2 Hz, 2H), 3.53 (q, J=7.0 Hz, 2H),7.19-7.22 (m, 1H), 7.26-7.29 (m, 1H), 7.30-7.35 (m, 2H), 8.16 (d, J=1.9Hz, 1H), 8.17 (d, J=1.9 Hz, 1H), 9.22 (t, J=5.5 Hz, 1H). ¹³C NMR (151MHz, DMSO) δ 34.0, 40.5, 81.4, 88.9, 116.2, 126.3, 127.5, 128.6, 130.2,133.0, 135.1, 141.6, 149.4, 159.8, 168.1. HRMS-ESI (m/z): [M+H]⁺ calcdfor C₁₅H₁₃ClI₂NO₂, 527.8719; found, 527. 8717.

N-(4-Chlorophenethyl)-2-hydroxy-3,5-diiodobenzamide (7h). Yield: 51%. ¹HNMR (500 MHz, DMSO-d₆) δ 2.85 (t, J=7.2 Hz, 2H). 3.51 (q, J=7.2 Hz, 2H),7.27 (d, J=8.5 Hz, 2H), 7.35 (d, J=8.5 Hz, 2H), 8.16 (d, J=1.9 Hz, 1H),8.18 (d, J=2.0 Hz, 1H), 9.21 (t, J=5.4 Hz, 1H). ¹³C NMR (151 MHz, DMSO)δ 33.7, 40.7, 81.4, 88.9, 116.2, 128.3, 130.6, 130.9, 135.1, 138.1,149.4, 159.8, 168.0. HRMS-ESI (m/z): [M+H]⁺ calcd for C₁₅H₁₃ClI₂NO₂,527.8719; found, 527. 8713.

N-(2,4-Dichlorophenethyl)-2-hydroxy-3,5-diiodobenzamide (7i). Yield:59%. ¹H NMR (500 MHz, DMSO-d₆) δ 2.97 (t, J=7.0 Hz, 2H), 3.53 (q, J=6.9Hz, 2H), 7.35-7.39 (m, 2H), 7.60 (d, J=1.1 Hz, 1H), 8.14-8.19 (m, 2H),9.23 (t, J=5.5 Hz, 1H). ¹³C NMR (151 MHz, DMSO) δ 31.8, 81.4, 88.8,116.2, 127.5, 128.7, 131.9, 132.5, 134.1, 135.1, 135.7, 149.4, 159.8,168.2. HRMS-ESI (m/z): [M+H]⁺ calcd for C₁₅H₁₂Cl₂I₂NO₂, 561.8329 found,561. 8319.

3,5-Dichloro-N-(4-(4-chlorophenoxy)phenyl)-2-hydroxybenzamide (8).Yield: 55%. ¹H NMR (600 MHz, CDCl₃) δ 6.96 (d, J=8.8 Hz, 2H), 7.04 (d,J=8.9 Hz, 2H), 7.31 (d, J=8.8 Hz, 2H), 7.50 (d, J=2.3 Hz, 1H), 7.53 (d,J=8.9 Hz, 2H), 7.56 (d, J=2.3 Hz, 1H), 8.02 (s, 1H), 12.16 (s, 1H). ¹³CNMR (151 MHz, CDCl₃) δ 116.6, 119.6, 120.3, 123.5, 123.7, 124.2, 124.6,128.8, 130.0, 131.6, 134.3, 154.9, 155.8, 156.0, 166.6. HRMS-ESI (m/z):[M+H]⁺ calcd for C₁₉H₁₃Cl₃NO₃, 407.9955; found, 407. 9955.

SYNTHETIC METHODS DOCUMENTS CITED

-   (1) Terada, H.; Goto, S.; Yamamoto, K.; Takeuchi, I.; Hamada, Y.;    Miyake, K. Biochim. Biophys. Acta 1988, 936, 504.-   (2) Gooyit, M.; Tricoche, N.; Lustigman, S.; Janda, K. D. J Med.    Chem. 2014, 57, 5792.-   (3) Gooyit, M.; Tricoche, N.; Javor, S.; Lustigman, S.; Janda, K. D.    ACS Med. Chem. Lett. 2015, 6, 339.-   (4) Gooyit, M.; Harris, T. L.; Tricoche, N., Javor, S., Lustigman,    S., Janda, K. D. ACS Infect. Dis. 2015, 1, 198.

1. A method of treatment of a Clostridium difficile infection in amammal, comprising administering to the mammal an effective dose of acompound of formula (I)

wherein X is halo or H, provided at least one X is halo, wherein thering bearing X is optionally further substituted with halo; wherein Aris benzyl, naphthyl, or indanyl, unsubstituted or independentlysubstituted with one or more halo, (C1-C4)alkyl, cyano, or nitro groups.2. The method of claim 1, wherein X is chloro or iodo.
 3. The method ofclaim 1, wherein Ar is substituted with halo or (C1-C4)alkyl, or both.4. (canceled)
 5. A compound having the formula: